Chrysanthemum stunt viroid newly reported in Slovenia

In August 2009, samples of chrysanthemum (Dendranthema × grandiflorum)were received for disease diagnosis from glasshouse crops growing in West Slovenia. Twelve out of 200 plants of cv. Miral, originating from Germany , showed viroid-like symptoms including stunting (Fig. 1) and yellow leaf spots (Fig. 2).Plants showing symptoms were distributed randomly in the glasshouse. ELISA tests of leaves with symptoms for Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus(INSV), Chrysanthemum stem necrosis virus (CSNV), Chrysanthemum B virus (CVB) and Tomato aspermy virus (TAV) were negative and examination by electron microscopy revealed no virus particles.

Leaf samples were also tested using reverse transcription-polymerase chain reaction (RT-PCR) with two sets of semi-universal pospiviroid primers: Pospi1-RE/FW and Vid-RE/FW (Verhoeven et al., 2004). With Pospi1-RE/FW primers, symptom-bearing chrysanthemum leaves yielded an amplicon of approximately 196 bp, whereas no product was obtained with Vid-RE/FW primers. PCR products were sequenced and a BLAST analysis of the sequenced amplicons revealed 100% identity with Chrysanthemum stunt viroid (CSVd) (GenBank Accession No. AB279769). Infection by CSVd was confirmed by RT-PCR using CSVd specific primers (Hooftman et al., 1996) and a product of the expected size (354 bp) was obtained. The PCR product was sequenced and subject to BLAST analysis. This sequence also shared 100% identity with the same isolate of CSVd (AB279769). Infectivity of the CSVd isolate from chrysanthemum was confirmed by mechanical inoculation on to tomato plants cv. Moneymaker in a greenhouse under quarantine conditions. Six weeks after inoculation the tomato plants were found to be infected with CSVd using RT-PCR testing. To our knowledge, this is the first report of CSVd in Slovenia .